First published online 18 January 2006
doi: 10.1242/dev.02225
Development 133, 631-640 (2006)
Published by The Company of Biologists 2006
XHas2 activity is required during somitogenesis and precursor cell migration in Xenopus development
Michela Ori1,
Martina Nardini1,
Paola Casini1,
Roberto Perris2,3 and
Irma Nardi1,*
1 Laboratori di Biologia Cellulare e dello Sviluppo, Dipartimento di Fisiologia
e Biochimica, Università di Pisa, Via Carducci 13, Ghezzano, Pisa (PI)
56010, Italy.
2 Dipartimento di Biologia Evolutiva e Funzionale, Università di Parma,
Viale delle Scienze 11/A Parma, (PR) 43100, Italy.
3 Divisione di Oncologia Sperimentale 2, Istituto Nazionale dei Tumori Aviano,
CRO-IRCCS, Via Pedemontana Occidentale, 12, Aviano (PN) 33081, Italy.
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Fig. 1. XHas2 is transcriptionally activated by activin.
(A,A') Ectopic XHas2 mRNA expression in
activin-injected embryos detected by whole-mount in situ
hybridization. The site of injection is visualized by the Red-gal staining
(red arrow). A, dorsal view; A', lateral view. (B) Comparison of
the expression profile of XBrachyury and XHas2 in animal
caps following activin treatment. XODC was used as positive control.
RT-indicates representative samples of RNA from activin-treated animal caps,
processed without reverse transcriptase and subsequently used as a negative
control for XHas2, XBrachyury and XODC amplification.
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Fig. 2. Myotome alterations in XHas2-Mo injected embryos. All
embryos are at stage 28-30 and are visualized by the expression of CA
(A,A') and by 12/101 immunoreactivity (B,B',C,D). Red-gal staining
identifies the injected side of the embryos. (A) Bending of the embryos
was observed in the injected side. (A') Example of a mild class
phenotype embryo; the myotome arrangement is altered in the injected side of
the embryo (arrow). (B) Lateral view of the control side of a
representative case of a severe class phenotype at stage 26 (arrowheads
indicate the somites). (B') Injected side of the embryo in B showing a
complete disruption of somites segmentation, as indicated by the arrowheads
and a strong reduction in the 12/101-positive cells (compare double-headed
arrows in B and B'). Red spots on the head and trunk region (Red-gal
staining) identify the site of injection. (C-D') Two representative
cases of severe class phenotype embryos, longitudinally sectioned and stained
with Hoechst to visualize the nuclei (C',D'). (C,D) The pattern of the 12/101
positive myocytes is altered within the somites in the injected side
(arrows).
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Fig. 3. Control experiments: stage 30 embryos were analyzed for CA
expression and HA distribution. (A) A representative embryo
injected with a control morpholino. (B-D) Embryos injected with 15 ng of
XHas2-Mo1 or with 15 ng of XHas2-Mo1 plus 800 pg
XHas2 mRNA (rescue experiment). The injected side of the embryo is
visualized by Red-gal staining. (B) Uninjected side of a
XHas2-Mo1-injected embryo and (C) injected side of the same
embryo showing the altered somites structure. (D) A representative case
of a rescue experiment in which the canonical somites structure is completely
recovered. (E) Ventral view of a wild-type embryo showing normal medial
fusion of the two halves of the heart anlage. (F) In
XHas2-Mo-injected embryos, heart formation is clearly impaired at the
level of the injected side (arrow). (G) Coronal section of stage 26
XHas2-Mo-injected embryos at the trunk level and double stained with
neurocan-GFP fusion protein (green) and Hoechst (blue). (H) High
magnification of the XHas2-Mo-injected side region indicated by the
white square in G; no HA detection is visible in the ECM surrounding the
myocytes. (I) High magnification of the control side region indicated
by the white square in G showing abundant HA in the ECM filling the myocytes
extracellular spaces. n, notochord; nt, neural tube.
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Fig. 4. XHas2 loss-of-function phenotype during early myogenesis.
Dorsal view of XHas2-Mo injected embryos at neurula stage analyzed
for XmyoD (A), CA (B,B',D) and p27
(C) expression by whole-mount in situ hybridization. Notably,
p27 expression appeared unaltered in primary neurons (arrow).
(B') Bisection of embryo shown in B. (D) Bisected XHas2-Mo
injected embryo at stage 22 showing a reduced somite mass in the injected
side. (E) Injected neurula stage embryo stained with PH3 antibody.
(F) Injected embryo analyzed by a BrdU incorporation assay (light blue,
X-gal staining). (G) Whole mount TUNEL staining in XHas2-Mo
injected neurula stage embryo. (G') Bisected neurula embryo
showing the increased number of apoptotic cells in the presomitic mesoderm of
the injected side (arrowheads). (H-I') Lateral view at
the level of trunk somitic region of XHas2-Mo injected embryos
processed by TUNEL assay. (H,I) Control side of stage 24 (H) and stage 30 (I)
embryos. (H',I') Injected side of the embryos shown in H and I. No
apoptotic cells were found at these stages in both control and injected side
of the embryos. (I'') In stage 30 embryos, physiological level of
apoptosis was found in the telencephalon (arrow).
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Fig. 5. XCD44 gene expression pattern. (A) XCD44
expression during Xenopus development analyzed by RT-PCR. (B)
Dorsal view of a stage 20 embryo showing XCD44 mRNA localization in
the presomitic mesoderm. (C) Lateral view of a stage 24 embryo:
XCD44 gene expression is detected in somites, cement gland
(arrowhead) and, at lower level, in the cranial NCC (arrows). (C')
Bisected embryo at stage 24 showing that in the trunk region XCD44 is
localized exclusively in the somites. The notochord (n) at this stage does not
express XCD44 mRNA. (D) Lateral view of stage 32 embryo:
XCD44 mRNA is present in somites, branchial arches (arrow) and in the
otic vesicle (arrowhead). A horizontal section at the level of the otic region
is shown in the inset. (E) Lateral view of a stage 37 embryo showing
the localization of XCD44 transcripts in the CNS (arrow), the dorsal
somite tips (black arrowhead), the notochord and migrating hypaxial muscle
cells (red arrowheads). (F-H) Dorsal view of XCD44-Mo injected
embryos at neurula stage, the injected side of the embryos is visualized by
the Red-gal staining. The expression of the following markers was detected by
whole mount in situ hybridization: (F) MyoD, (G) p27 and (H)
CA. (I) An example of a XCD44-Mo injected embryo
processed by TUNEL assay. The apoptotic cells are in blue; red spots indicate
the site of injection.
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Fig. 6. Analysis of hypaxial cell migration in XHas2-Mo and
XCD44-Mo injected embryos. (A,A') Lateral view of
control (A) and injected (A') side of an XHas2-Mo injected
embryo (mild phenotype) at stage 37 showing the relative position of
differentiating hypaxial muscle cells, as highlighted by 12/101 antibody
staining (arrows). (B,B') Transversally sectioned XHas2-Mo
injected embryo hybridized with CA and coloured with Hoechst to
visualize the nuclei, in which it is evident that the somite structure is
altered and the ventrolateral migration of hypaxial muscle cells is impaired
(red arrow). (C) Lateral view of the control side of a
XHas2-Mo-injected embryo analyzed for XPax3 expression by in
situ hybridization at stage 37 highlighting hypaxial muscle cells migrating
ventrally (arrow). (C') Injected side of the embryo shown in C,
in which hypaxial muscle cells are blocked at the ventral aspect of somites
(red arrow). (D,D') Stage 37 XCD44-Mo injected embryos
immunostained with the 12/101 antibody. Although the trunk musculature appears
unaffected by XCD44 loss, migration of hypaxial cells is greatly
reduced. (D) Control side of the embryo; (D') injected side. (E-G)
Ventral views of stage 43 embryos immunostained with the 12/101 antibody that
show the final position of the ventral body wall musculature in uninjected
(E), XCD44-Mo injected embryo (F) and XHas2-Mo injected
embryo (G). The side of injection is visualized by X-gal staining in blue and
indicated by the arrow. (E'-G') Lateral views of the
same embryos shown in E-G, respectively.
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Fig. 7. Stage 32 XHas2-Mo and XCD44-Mo injected embryos
analyzed for XGremlin expression by whole-mount in situ
hybridization. (A) Lateral view of the control side of two embryos,
showing NCC migration pathways. (A') Lateral view of the injected
side, revealed by Red-gal staining, of the embryos shown in A: no defined NCC
migration pathways are recognizable. (B) Higher magnification of one of
the embryo showed in A. (B') Injected side of the embryo shown in
B; NCCs are still present but they do not follow their normal migration
routes. (C) Longitudinal section of an XHas2-Mo injected
embryo: trunk NCCs appear dispersed along the somite external aspect (injected
side, red arrowheads) instead of migrating along the intersomitic boundaries
(control side, yellow arrowheads). (D,D') Double-labelling of
stage 32 embryo for XGremlin (blue) and 12/101 muscle-specific
antigen (orange), whereas red staining reveals the injected side of the
embryo. From the comparison of the control (D) and the injected sides (D'), it
seems that the impairment of trunk NCC migration parallels the reduction and
developmental alterations of somites. (E,E') Stage 32 XCD44-Mo
injected embryos analyzed for XGremlin expression. No differences in
NCC migration are discernible between the control side (E) and the injected
side (E') of the embryo.
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